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1
Weihong Pan, M.D., Ph.D.
Pennington Biomedical Research Center
Baton Rouge, LA
The Blood-Brain and Blood-CSF Barriers
Principles o! Pharmacolog", #ulane Med $%%&
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Course objectives
#he lood'C() and lood'rain arriers as
d"namic regulator" inter!aces
#he morphological*cellular asis andsustrates o! the arriers
Anal"tical methods
)unctional implications in normal andpathological conditions
Pharmacological manipulations
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1+%% Lewandows-" Prussian
lue
1+%$ hrlich /ital staining
with man" d"es1+%+ 0oldmann tr"pan lue
i
1+12 0oldmann tr"pan lue
into C()
1+3% #schirgi d"e'proteincomple4
1+5%6s peptides cross the
BBB
$%%%6s lood'rain regulator"
inter!ace
Short history
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Cerebrospinal fluid (CSF)
Production !loor o! the lateralentricle and third entricle, "choroid ple4us
Circulation L./. '7 888./. '7 8/./.'7 e4it entricular s"stem intoarious asal cisterns and thento suarachroid space
Drain ac- to lood iaarachnoid granulation to superiorsagittal sinus, or ia spinal nereroots, or ia ol!actor" tracts
)rom 9ohanson C
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5
CSF volume in human based on
NMR imaging (Kohn 1991)
#otal 1&:.3 ; :
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Function of CSF
Maintenance o! a constant e4ternal
enironment !or neurons and glia
Mechanical cushion to protect the rain anduo"ant to the hea" rain =1:%% g>
(ere as a l"mphatic s"stem and a conduit
!or neuropeptides p? o! C() regulates pulmonar" entilation
and CB)
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Cerebral Edema
/asogenic edema A state o! increased
e4tracellular !luid olume Brain tumor, ascess, in!arct,hemorrhage Glucocorticoids, hypertonic solutions
C"toto4ic edema #he swelling o! cellular
elements ?"po4ia*asph"4ia, water into4ication, meningitis,encephalitis, Re"e6s s"ndrome
8nterstitial edema Attriuted to increased waterand sodium in perientricular white matterstructie h"drocephalus Surgical shunting, acetazolamide
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Hydrocephalus
structie h"drocephalus Congenital mal!ormations A!ter in!lammation or hemorrhage Mass lesions
ormal pressure h"drocephalus Communicating h"drocephalus
erproduction o! C() De!ectie asorption o! C() /enous drainage insu!!icienc"
?"drocepalus e4 acuo =rain atroph">
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CSF sampling
Contraindiation or !reaution in" #$C%
Pressure !rom lumar puncture &3'1+3 mm C() =?$>, or
3 13 mm?g
Cells % " in!lammation, tumor, or other cereraldamage
Protein E 23 mg*dl in loc-age or increased BBB
permeailit". M( shows higher F'gloulin and oligoclonal
ands 0lucose 7 :% mg*dl G in meningeal tumor, !ungal or #B
in!ection, sarcoidosis
Appearance not cloud" or 4anthochromia
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Blood'C() Barrier
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)luid compartments o! the lood'rain'C() s"stem
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Blood-Brain Barrier (BBB) and Blood-
Spinal Cord Barrier (BSCB) Barrier and regulator" unit etween Hcapillaries
and postcapillar" enulesI J H4tracellular
compartment o! rain and spinal cordI Composed o! ascular endothelia, their
asement memrane, peric"tes, and glial
periascular !eet =the end!eet>
#ight Kunctions in'etween endothelial cells
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13
)rom Dais #P
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CD21 immunostaining showing lood essels
and prealence o! the B(CB
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Put B(CB into the conte4t
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olarity
Distinctie apical and asolateral sur!aces
Di!!erent distriution o! transporters
Directional transport
Resolution in in'itro models 2'D or :'D
cultures
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ermeability of a membrane
and !inetic analysis 8ssues related to mechanisms o! penetration
simple di!!usion, carrier'mediated transport,
and actie transport
(tead"'state distriutions
!!ects o! lipid soluilit" and !low
Multiple compartments and regionaldi!!erences
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"inetic aspects of the BBB
Permeability the characteristics o! a
memrane, or a group o! memranes,
separating two !luids
Permeability coefficient how rapidl" a giensustance will permeate the memrane
dM*dt %A=Cpl CBr=W>>
Transfer coefficient PA*/wW P(*/wstimated !raction o! rain weight /w %.13 !ore4tracellular !luid onl" and %.
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#athematical treatment
Crone6s single'pass techniOue =1+&%6s> A'/ di!!erenceo! test and re!erence sustances
Brain upta-e inde4 =ldendor!, 1+ re!erence
sustance =2?$, or 1:C'utanol with P(*) o! 1. hasrapid, !low'dependent permeation to compensate !orCB) ariations
nidirectional !lu4 in rapidl" eOuilirating space Patla-'
Blaserg')enstermacher treatment =1+5%6s> (lowl" penetrating compounds that are stale in
circulation Ban-s'astin multiple'time regressionanal"sis =1+5%6s>
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Cerebral blood flo$
)ic- principle dCi )=Ca'C>
8ndicator !ractionation techniOue with 1:C'
utanol or 1:C'iodoantip"rine Quantitatie autoradiograph" !or regional
di!!erences
Cereral lood olume measured " 31Cr'RBC or 11C inhalation
and positron emission tomograph" =P#>
4tracellular space inulin space
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%isruption of the BBB
#rauma mechanical, irradiation, chemical
8n!lammation
8schemia *hemorrhage #umor
Dem"elinating disease
eurodegeneration Certain metaolic and congenital disorders
pileps"
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EBA SF Overlay
Left
hemisphere
Right
hemisphere25 m
Demonstration o! BBB disruption " an6s lue alumin
and sodium !luorescein sample !rom mouse rain
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&smotic opening of the BBB
A therapeutic approach
What are the osmotic agents
' mannitol, RMP'< =rad"-inin receptor agonist>, etc. ?as een used clinicall" in C( tumor chemotherap"
Question:
What are other ways to deliver agents to the CNS?- viral vectors fusion !roteins carrier antibodies
immunoli!osomes
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8ssue o! circumentricular organs =C/>
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'*-eptide
#ermination
o! actions
#ransc"tosis 'transport
8nteractionswith othere!!ectormolecules
!!ects oncells
Bindinginteractions
Receptordistriution8ntracellular
degradation
Degradation
4tracellular
matri4on'speci!icassociation
/olume o!
distriution
In blooIn bloo At the barrierAt the barrier InsieInsie!"S!"S
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+ransport of substances across the
BBB "lood-to-CNS
influ#
"rain-to-blood
efflu#
8ons
Amino Acids and organic acids
0lucose and other caroh"drates Biogenic amines
ucleotide precursors
Peptides, proteins and lipoproteins
(teroid and th"roid hormones /itamins, trace metals
Chemotherap" agents, antiiotics
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, specific eample. C/S chemotherapy
http://www.jci.org/cgi/content/full/116/3/642/F1 -
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Chemotheraphy to the C/S
("stemic #4 not routine since most agents hae
limited penetration despite partiall" disrupted BBB
8ntrathecal therap" is inasie and con!ined to onl"
three agents methotre4ate, c"tosine arainoside,
thiotepa
niOue side'e!!ects aseptic meningitis =:2S>,
chemotherap"'related leu-oencephalopath"
ewer approaches include gene therap" =with iral
ectors>, osmotic opening o! the BBB, and c"to-ines
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%rug resistance remains one of the primarycauses of suboptimal outcomes in cancer therapy
A#P'inding cassette =ABC> transporters
A#P'dependent drug e!!lu4 pumps,oere4pression in chemo'resistant tumorsand upregulation with disease progression
A large !amil", e.g., P'gp, MRP1, ABC0$
8nhiitors e!!ectie in animal studies hae notdemonstrated clinical e!!icac"
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http://theoncologist.alphamedpress.org/cgi/content/full/8/5/411/F1 -
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Another e4ample C"to-ine transc"tosis across the BBB
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&h' is it im!ortant to stud' the interations&h' is it im!ortant to stud' the interationso 'toines *ith the BBB+o 'toines *ith the BBB+
Peripheral c"to-ines modulateneuroendorine untionsin the C(
Peripheral c"to-ines a!!ect CNS regeneration C"to-ines in the rain and circulation
in!luence !eeding ehaior, sleep, mood,cognition, and a lot o! other important C(actiities
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0hat $e focus in our laboratory(teaching /europharm is just my side job)
)unctional implications o! c"to-ine transport
across the BBB
#ra!!ic-ing o! c"to-ines within cereral
microessel endothelial cells
Regulator" !actors !or c"to-ine transport and
targeted drug delier"
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Studies with Anesthetized mice:
Eample '. multiple-time regression analysis
?"pothesis in!lu4 is linear during stud" interal
Design i olus, di!!erent time o! circulation,each mouse represents one time point
Controls ascular mar-ers stailit" studies
compartmental distriution in'situ per!usion
Data anal"sis e4posure time, in!lu4 trans!ercoe!!icient, Supta-e
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% : & 5 1%2.3
:.%
:.3
3.%
#ime =min>
Log
=serum
radioacti1it">
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% 3 1% 13 $% $3 2% 23 :%
%
1%%%
$%%%
2%%%
:%%%
3%%%
&%%%
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parench"ma,no per!usion parench"ma, per!used capillar", no per!usion capil lar", per!used%
1
$
2
:
3
&
The bottom line? The behavior of itsinteractions with the """ may determine the
CNS effects &or side-effects' of a com!ound(
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SC*. ,ll in due course
)mmediately after lumbar contusion:suarachnoid hemorrhage petechial hemorrhages in gra" and white matter !irinoidnecrosis o! essels
*+ min - , h after traumanecrotic essels, aneur"smal dilitation, periascular hemorrhage, neuronal and glial
necrosis
- * d after traumapol"morphonuclear leu-oc"te and macrophage in!iltration a4onal spheroids
w% - m after traumareduced in!lammator" changesc"stic changes and gliosis
Balentine 9.D.,Balentine 9.D., .ab/ )nvest.ab/ )nvest. 1+
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%ynamic changes of the BSCB
after SC* Reersile
Biphasic Correlation with seerit" o! inKur"
(el!'repair !unction o! the B(CB is also
re!lected " the regulator" changes o!speci!ic transport s"stems !or c"to-ines
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Brain #) Brain Alumin (pinal cord #) (pinal cord Alumin%
1%
$%
2%
1 w- e4cess #)
NNN NNNN
1 w-
control
NNN NNNN
Right L1 hemisection 8ncreased #)upta-e in all C( regions is still saturale.
Tissue/s
erumR
atio
oRadioativit'(l/g)
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Brain Cerical #horacic Lumar (pinal cord%
3
1%
13
W# cont
W# (C8
DMA contDMA (C8
NNN NNN
NN NN NNN
N
++#c'Alumin#issue*(erum
Ratioo!Radioac
ti1it"=
l*g>
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Brain Cerical #horacic Lumar (pinal cord%
3
1%
13
$%
$3
W# cont
W# (C8
DMA cont
DMA (C8
NNN
NNN
NNN
1$38'#B)#issue*(erum
Ratioo!Ra
dioactiit"=
l*g>
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% 1% $% 2% :% 3% &%%
$
:
&
5
1%
D apical to asolateral
cont apical to asolateral
D asolateral to apical
cont asolaterl to apical
#ime =min>
1$38'
#B
)
R
atio
=4
1%
2>
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nderstand and modulate the transport
s"stems Modulate general permeailit"
Design analogs !or etter penetration
se ehicles e.g., iral ectors, liposomes B"pass the BBB
0hat $e can do to improve C/S drug
delivery
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Summary
0emember #he BBB*B(CB as a d"namic
regulator" inter!ace
0emember #he morphological*cellular asisand sustrates o! the arrier
1#!osure to Anal"tical methods
2nderstand )unctional implications in normal
and pathological conditions
Thin% about Pharmacological manipulations
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#emory test Concepts ' "es Applications ' ma"e #hin- o! the BBB in case o! drug delier" to the C(
Re!erences =supplied !or "our interest, not !or !inal e4am>Begle" and Brightman, (tructural and !unctional aspects o! the lood'rain arrier. Prog.
Drug Res., $%%2
Pan and astin, Wh" stud" transport o! peptides and proteins at the neuroascularinter!ace. Brain Res Re, $%%:
9ohanson C et al., nhanced prospects !or drug delier" and rain targeting " thechoroid ple4us'C() route. Pharm. Res., $%%3
Boo-s
Dason and (egal, Ph"siolog" o! the C() and lood'rain arriers. CRC Press.
(harma and Westman, Blood'spinal cord and rain arriers in health and disease. lseier.
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Blood'Brain Barrier 0roup
L:%2%, Pennington Biomedical Research Center
Baton Rouge, LA